Methods for forming a nanopore in a lipid bilayer

ABSTRACT

A method of forming a nanopore in a lipid bilayer is disclosed. A nanopore forming solution is deposited over a lipid bilayer. The nanopore forming solution has a concentration level and a corresponding activity level of pore molecules such that nanopores are substantially not formed un-stimulated in the lipid bilayer. Formation of a nanopore in the lipid bilayer is initiated by applying an agitation stimulus level to the lipid bilayer. In some embodiments, the concentration level and the corresponding activity level of pore molecules are at levels such that less than 30 percent of a plurality of available lipid bilayers have nanopores formed un-stimulated therein.

CROSS REFERENCE TO OTHER APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/445,180, filed Aug. 16, 2021, which is a divisional application ofU.S. patent application Ser. No. 16/516,166, entitled SYSTEMS FORFORMING A NANOPORE IN A MEMBRANE, filed Jul. 18, 2019, now U.S. Pat. No.11,092,589, which is a continuation of U.S. patent application Ser. No.15/488,432, entitled SYSTEMS AND METHODS FOR FORMING A NANOPORE IN ALIPID BILAYER, filed Apr. 14, 2017, which is a continuation of U.S.patent application Ser. No. 14/150,322, now U.S. Pat. No. 9,678,055,entitled METHODS FOR FORMING A NANOPORE IN A LIPID BILAYER, filed Jan.8, 2014, which is continuation in part of U.S. patent application Ser.No. 12/658,591, now U.S. Pat. No. 9,605,307, entitled SYSTEMS ANDMETHODS FOR FORMING A NANOPORE IN A LIPID BILAYER, filed Feb. 8, 2010,each of which are incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

Nanopore membrane devices having pore size in the order of 1 nanometerin internal diameter have shown promise in rapid nucleotide sequencing.When a voltage potential is applied across the nanopore immersed in aconducting fluid, a small ion current due to conduction of ions acrossthe nanopore can be observed. The size of the current is sensitive tothe pore size. When a molecule such as a DNA or RNA molecule passesthrough the nanopore, it can partially or completely block the nanopore,causing a change in the magnitude of the current through the nanopore.It has been shown that the ionic current blockade can be correlated withthe base pair sequence of the DNA molecule.

However, this technology still faces various challenges and so far ithas not been able to discriminate down to a single base pair. Inparticular, the electrical potential needed to attract a ssDNA moleculein the nanopore tends to cause the ssDNA molecule to pass through thenanopore very quickly, making analysis difficult. To solve this problem,attempts have been made to tether the ssDNA to a bead to arrest themovement of the ssDNA molecule through the nanopore. However, such anapproach may involve extensive sample preparation and may not besuitable for small sample sizes. Improved techniques for DNA analysisusing nanopore membrane devices are needed.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the invention are disclosed in the followingdetailed description and the accompanying drawings. Note that thefigures are intended to illustrate the various embodiments of thepresent invention and they are not necessarily drawn to scale.

FIG. 1 is a schematic diagram of an embodiment of a nanopore devicecomprising a nanopore-containing a lipid bilayer.

FIG. 2 is a schematic diagram of an embodiment of a circuit used in ananopore device for controlling an electrical stimulus and for detectingelectrical signatures of an analyte molecule.

FIG. 3A is a perspective view of a schematic diagram of an embodiment ofa chip that includes a nanopore device array.

FIG. 3B is a cross sectional view of the chip shown in FIG. 3A.

FIG. 4A is a schematic diagram depicting an embodiment of a process forforming a lipid bilayer on a solid substrate.

FIG. 4B illustrates phase I of the nanopore device 100 during process400.

FIG. 4C illustrates phase II of the nanopore device 100 during process400.

FIG. 4D illustrates phase III of the nanopore device 100 during process400.

FIG. 5A is a schematic diagram of an embodiment of a process forinserting a nanopore into a lipid bilayer.

FIG. 5B illustrates phase III of the nanopore device 100 during process500.

FIG. 5C illustrates phase IV of the nanopore device 100 during process500.

FIG. 5D illustrates phase V of the nanopore device 100 during process500.

FIG. 5E illustrates phase I of the nanopore device 100 during process500.

FIG. 6A is a schematic diagram illustrating an embodiment of a processfor manipulating, detecting, characterizing, correlating, analyzingand/or sequencing a molecule in a nanopore.

FIG. 6B illustrates phase IV of the nanopore device during process 600.

FIG. 6C illustrates phase V of the nanopore device during process 600.

FIG. 7A illustrates an embodiment of a progression electrical stimulus.

FIG. 7B illustrates an embodiment of a progression electrical stimulus.

FIG. 7C illustrates an embodiment of a progression electrical stimulus.

FIG. 7D illustrates an embodiment of a progression electrical stimulus.

FIG. 8A is a schematic diagram illustrating an embodiment of a processfor reversing the progression of a molecule in a nanopore.

FIG. 8B illustrates phase V during process 800.

FIG. 8C illustrates phase VI during process 800.

FIG. 9 is an embodiment of a resistance profile of a molecule driventhrough the nanopore.

DETAILED DESCRIPTION

The invention can be implemented in numerous ways, including as aprocess; an apparatus; a system; a composition of matter; a computerprogram product embodied on a computer readable storage medium; and/or aprocessor, such as a processor configured to execute instructions storedon and/or provided by a memory coupled to the processor. In thisspecification, these implementations, or any other form that theinvention may take, may be referred to as techniques. In general, theorder of the steps of disclosed processes may be altered within thescope of the invention. Unless stated otherwise, a component such as aprocessor or a memory described as being configured to perform a taskmay be implemented as a general component that is temporarily configuredto perform the task at a given time or a specific component that ismanufactured to perform the task. As used herein, the term ‘processor’refers to one or more devices, circuits, and/or processing coresconfigured to process data, such as computer program instructions.

A detailed description of one or more embodiments of the invention isprovided below along with accompanying figures that illustrate theprinciples of the invention. The invention is described in connectionwith such embodiments, but the invention is not limited to anyembodiment. The scope of the invention is limited only by the claims,and the invention encompasses numerous alternatives, modifications andequivalents. Numerous specific details are set forth in the followingdescription in order to provide a thorough understanding of theinvention. These details are provided for the purpose of example and theinvention may be practiced according to the claims without some or allof these specific details. For the purpose of clarity, technicalmaterial that is known in the technical fields related to the inventionhas not been described in detail so that the invention is notunnecessarily obscured.

Techniques for manipulating, detecting, characterizing, correlatingand/or determining a molecule using a nanopore device are describedherein. In one example, an acquiring electrical stimulus is appliedacross a nanopore-containing lipid bilayer characterized by a resistanceand capacitance, where the acquiring electrical stimulus is of a levelthat tends to draw the molecule from a surrounding fluid into thenanopore. A change is detected in the electrical characteristics of thelipid bilayer resulting from the acquisition of at least a portion ofthe molecule into the nanopore. In response, the electrical stimuluslevel is changed to a holding electrical stimulus level. Typically, thelevel of the acquiring electrical stimulus that tends to draw a moleculefrom a surrounding fluid into the nanopore also tends to cause themolecule to progress through the nanopore too quickly. In order to trapthe molecule in the nanopore for further detailed characterization, theelectrical stimulus level often needs to be quickly reduced to a lowerholding electrical stimulus level after detecting a change in theelectrical characteristics of the nanopore containing lipid bilayerresulting from the acquisition of at least a portion of the moleculeinto the nanopore.

After the molecule is trapped in the nanopore, a progression electricalstimulus (e.g., a variable electrical stimulus) is then applied acrossthe nanopore-containing lipid bilayer until the molecule progressesthrough the nanopore. The progression electrical stimulus level is suchthat it allows the molecule to progress through the nanopore in afashion that allows recording of useful electrical signature(s) of themolecule for characterization. In some embodiments, the progressionelectrical stimulus level is lower than that of the acquiring electricalstimulus and higher than that of the holding electrical stimulus. As themolecule progresses through the nanopore, one or more electricalsignature(s) of the molecule is recorded. The molecule can then becharacterized based on the detected electrical signature(s).

A reverse progression electrical stimulus may also be applied to allowthe molecule to reverse progress or rewind through the nanopore. Thereverse progression electrical stimulus may be applied before, afterand/or interspersed with the progression electrical stimuli. By cyclingthe progression electrical stimuli and the reverse progressionelectrical stimuli, repeat measurements of the molecule can be obtainedduring molecule progression and/or reverse progression through thenanopore. In some embodiments, the cycling is applied to a selectedregion of the molecule, such as a SNP site, a copy number variationsite, a methylated site, a protein binding site, an enzyme binding site,a repetitive sequence site, and a restriction enzyme site to allow finermeasurements, and better accuracy for the selected region of themolecule. In one example, a progression electrical stimulus may beapplied first, followed by a reverse progression electrical stimulus,which is then followed by another progression electrical stimulus. Byrepeating measurements for the same portion of a molecule, an improvedsignal to noise ratio for measurements can be achieved. In one example,a plurality of reverse progression electrical stimuli is interspersedwith a plurality of progression electrical stimuli, where each of theplurality of progression electrical stimuli is followed by a reverseprogression electrical stimulus. In some embodiments, the polarity ofthe reverse electrical stimulus level is reversed compared to theprogression electrical stimulus, and the reverse electrical stimuluspulls the molecule in a reverse progression direction. In someembodiments, the reverse electrical stimulus has the same polarity but asmaller magnitude (or a magnitude of zero) compared to the progressionelectrical stimulus and the natural tendency of the molecule to reverseprogress through the nanopore pulls the molecule in the reverseprogression direction. In such cases, the reverse electrical stimulusmay serve to slow down the reverse progression of the molecule throughthe nanopore. The electrical signature(s) detected during the reverseprogress can also be used to characterize the molecule. Under certaincircumstances, the molecule can move in a more predictable and/or slowerspeed when it reverse progresses through the nanopore and the electricalsignature(s) recorded may have better quality and signal to noise ratio.In one example, the molecule being characterized is a dsDNA molecule andwhen a reverse progression electrical stimulus is applied, the unzippedssDNA molecules re-anneal to form a dsDNA molecule as it reverseprogresses through the nanopore. In this example, the reverseprogression electrical stimulus has the same polarity but a smallermagnitude than the progression electrical stimulus. The natural tendencyof the unzipped ssDNA molecules to re-anneal to form a dsDNA moleculedrives the molecule in the reverse progression direction. The reverseprogression electrical stimulus acts to slow down the speed at which theDNA molecule reverse progresses through the nanopore. In the case wherethe reverse progression electrical stimulus has the same polarity as theprogression electrical stimulus, an increase in the magnitude of thereverse progression electrical stimulus slows down the reverseprogression of the molecule. In the case where the reverse progressionelectrical stimulus has the opposite polarity as the progressionelectrical stimulus, an increase in the magnitude of the reverseprogression electrical stimulus speeds up the reverse progression of themolecule. In the example where the ssDNA re-anneal to form a dsDNA asthe DNA molecule reverse progresses through the nanopore, the tendencyfor the ssDNA molecules to re-anneal to form the dsDNA (e.g., the energyreleased when the ssDNA molecules re-anneal to form the dsDNA) mayaffect the polarity and/or the magnitude of the reverse progressionelectrical stimulus. In other examples where a molecule re-hybridizewith a hybridization marker as the molecule reverse progresses throughthe nanopore, the tendency for the molecule to re-hybridize with thehybridization marker (e.g., the energy released when the moleculere-hybridize with the hybridization marker) may affect the polarityand/or the magnitude of the reverse progression electrical stimulus.

The molecule being characterized using the techniques described hereincan be of various types, including charged or polar molecules such ascharged or polar polymeric molecules. Specific examples includeribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules. TheDNA can be a single-strand DNA (ssDNA) or a double-strand DNA (dsDNA)molecule. Other examples include polypeptide chain or protein.

The molecule can be modified prior to analysis. For example, themolecule can be hybridized with a hybridization marker prior toanalysis. The hybridization marker may be anything that can bind to themolecule being characterized. The hybridization marker may serve tomodify the energy (e.g., voltage level) required to move the moleculethrough the nanopore and/or may change the electrical signature of themolecule as it is threaded through the nanopore, by for exampleaffecting the conformation of the molecule being characterized, theenergy required to tear the molecule being characterized apart from thehybridization marker in order to thread the molecule through thenanopore, the energy released when the molecule is rehybridized with thehybridization marker. It should be noted that the hybridization markermay or may not necessarily move through the nanopore with the moleculebeing characterized. Examples of the hybridization marker include DNA,RNA, modified DNA, modified RNA, ligand, polymer, vitamin, fluorescentmolecule, beads. For example, in cases where the molecule beingcharacterized comprises a nucleotide molecule (e.g., DNA molecule), thehybridization marker can include a strand of nucleotide sequence (e.g.,DNA or RNA sequence) or modified nucleotide sequence (e.g., modified DNAor RNA sequence) that complements the entire nucleotide molecule beingcharacterized or a region of interest of the nucleotide molecule beingcharacterized. The hybridization marker can for example include anucleotide sequence that complements the nucleotide sequence of asingle-nucleotide polymorphism (SNP) site, a copy number variation site,a methylated site, a protein binding site, an enzyme binding site, arepetitive sequence site, a restriction enzyme site, miRNA site, siRNAsite, tRNA site, a transposon site, a centromere site, a telomere site,a translocation site, an insertion site, or a deletion site.

The electrical stimulus described herein can be various electricalstimuli, such as an applied current and an applied voltage. The currentcan be a direct current (DC) and/or an alternating current (AC). Theelectrical stimulus can constitute a series of electrical pulses.

The electrical signature may include any measurable electrical propertyof the nanopore, lipid bilayer, or nanopore-lipid bilayer system thatchanges as the molecule progresses through the nanopore that isindicative of the molecule's properties or structure. For example,different individual base pairs of a DNA molecule or sequences of basepairs may cause the nanopore to have different ionic current flow orresistance. Also, more or less voltage may be required to move a trappedDNA molecule through the nanopore because of different bonding strengthbetween different base pairs of the DNA molecule. The bonding strengthbetween different base pairs of the DNA molecule can be made larger orsmaller by hybridizing the DNA molecule to different hybridizationmarker. Therefore, in various embodiments, the electrical signature mayinclude instantaneous measurements or measurements made over time ofvoltage, resistance, and/or current profile across the lipid bilayer.For example, the electrical signature may include the magnitude(s) ofthe variable electrical stimulus required to affect the progression ofthe molecule through the nanopore. The electrical signature may also bea combined electrical signature combining electrical signatures ofvarious discrete portions or frames of the molecule as it progressesthrough the nanopore. For example, characterizing the DNA molecule maybe based on a combined electrical signature combining electricalsignatures for various frames of the DNA molecule, each framecorresponding to an electrical signature of region of the DNA molecule(e.g., 1 to 20 base sequence) as the molecule threads through thenanopore under an applied electrical stimulus. In some embodiments,electrical signatures of one or more overlapping frames of a moleculemay be combined and deconvolved to produce the electrical signature ofthe molecule. Overlapping the sampling frames may allow for a moreaccurate characterization of the molecule.

In some embodiments, in order to gather more data that may be used tocharacterize a molecule, multiple electrical measurements of themolecule may be acquired under the same or different chemical orenvironmental conditions. Multiple electrical measurements of the samemolecule may be achieved by repeatedly rewinding the molecule throughthe nanopore and repeating the electrical measurements under the same ordifferent conditions. In some embodiments, different chemical orenvironment conditions may be achieved by varying one or more of variousenvironmental variables, such as pH, salt concentration, glycerolconcentration, urea concentration, betaine concentration, formamideconcentration, temperature, divalent cation concentration, and otherenvironmental variables. The repeat measurements can be carried out in asingle experiment to the same molecule or in different experiments tothe same molecule or different molecules. The repeat measurements may becarried out by rewinding the molecule in the nanopore under an appliedreverse progression electrical stimulus. In some embodiments, the repeatmeasurements may be carried out for one or more regions of interest ofthe molecule, such as single nucleotide polymorphism (SNP) sites andmethylated sites of a DNA molecule. In some embodiments, the moleculebeing characterized may assume different conformations and/ororientations as it is drawn through the nanopore, causing the measuredelectrical signature(s) of the same molecule to differ from experimentto experiment and making it difficult to characterize the molecule. Byrepeatedly measuring the electrical signature(s) of the same molecule,usually under the same conditions, and obtaining a library of uniqueelectrical signatures of the molecule from the repeat measurements, thedifferent signatures from the different conformations and/ororientations of the molecule can be used to cross-check and increase theconfidence in identifying a particular biomarker.

Characterization of the molecule can include determining any property ofthe molecule that causes a variance in a measurable electricalsignature. For example, a base sequence of an DNA molecule may bederived from measuring a variance in ionic current flow (or electricalresistance) through the nanopore as the DNA molecule progresses throughthe nanopore, and/or from measuring the voltage required to pull atleast a portion of the molecule (e.g., a single strand of a dsDNAmolecule) through the nanopore at various points of the molecule. If themolecule being characterized is a dsDNA, characterizing the molecule mayinclude identifying one or more GC and/or AT base pairs of the dsDNAmolecule. Characterization of the molecule can also include determininga property of the molecule by comparing and correlating the measuredelectrical signature(s) of the molecule with electrical signature(s) ofknown molecules to obtain a possible structure of the molecule. Forexample, the base sequence of a segment of a DNA molecule can bedetermined by comparing and correlating the measured electricalsignature(s) of the DNA molecule with electrical signature(s) of knownDNA segments. In some embodiments, the molecules being characterized areDNA segments of a gene. The sequences of the DNA segments determinedusing the techniques described herein can be used for de novo sequencingof the gene. In one example, the gene being sequence may be fragmentedinto shorter nucleotide sequences (e.g., 50 to 10,000 base pairs) usingone or more restriction enzymes. Sequences of individual DNA segmentsmay be determined by correlating the detected electrical signature(s) ofthe DNA segment with that of known DNA sequences. The entire sequence ofthe genome can then be reconstructed by aligning overlapping portions ofthe fragmented DNA segments.

The herein described techniques for manipulating and characterizing amolecule may be highly sensitive and may not require extensive sampletreatment, such as amplification, separation, and derivatization, thusvery small amount of sample may be needed. This makes the techniquesdescribed herein especially suitable for applications that require highsensitivity and/or offer limited sample size. Examples of suchapplications include cancer biomarker screening, infectious diseasedetection, newborn screening, and bioterrorism agent screening.

Additionally, techniques for assembling a lipid bilayer on asubstantially planar solid surface are described herein. The lipidbilayer compatible surface may be isolated by one or more lipid bilayerincompatible surfaces that are not suitable for forming a lipid bilayer.The lipid bilayer incompatible surfaces may limit the size of the lipidbilayer formed to the edges of the lipid bilayer compatible surfacessince the lipid bilayer only forms on lipid bilayer compatible surfacesand does not form on lipid bilayer incompatible surfaces. In oneexample, a lipid suspension (e.g., aqueous electrolyte solutioncontaining suspended lipid colloids) is deposited over the lipid bilayercompatible surface as well as the adjacent lipid bilayer incompatiblesurfaces. In some embodiments, the lipid bilayer compatible surfacecomprises a hydrophilic material. Any materials that tend to allowformation of a lipid bilayer may be used. In some embodiments, the lipidbilayer incompatible surface comprises a lipophilic material. Anymaterials that tend to inhibit formation of a lipid bilayer may be used.A bubble of lipids filled with fast diffusing gas molecules is thenformed on the lipid bilayer compatible surface. The bubble is hereintermed a lipid bilayer initiating bubble. The gas molecules are allowedto diffuse out of the bubble and the bubble folds or collapses to form alipid bilayer on the solid surface.

Various techniques may be used to form the lipid bilayer initiatingbubble described above. For example, the lipid suspension deposited onthe lipid bilayer compatible surface (e.g., electrode surface) mayinclude chemicals that can react or decompose to form fast diffusing gasmolecules. Fast diffusing gas molecules can be any gaseous moleculesthat can diffuse quickly through lipid layers. In general, largermolecules or ionic gaseous molecules do not diffuse very well throughthe lipid bilayer, while smaller nonpolar molecules can diffuse rapidlythrough the lipid bilayer. Examples of fast diffusing gaseous moleculesinclude O₂ and CO₂. In one example, the lipid suspension includespotassium formate molecules and an bubble initiating electrical stimulushaving a range of 0.3 V to 3.0 V is applied to the lipid suspension for100 ms to 1 s to cause the formate molecules to decompose to form fastdiffusing C₂O. In another example, a bubble initiating electricalstimulus having a range of 0.5 V to 3.0 V may be applied to a lipidsuspension to oxidize H₂O to form fast diffusing O₂ gas molecules.

The structural integrity and/or the electrical characteristics of thelipid bilayer may be examined using various techniques to make sure ithas the necessary structural and/or electrical characteristics. In oneexample, an alternating current (AC) may be applied across the lipidbilayer to detect the capacitance of the lipid bilayer. In someembodiments, if the detected capacitance is greater than approximately 5fF/μm², the lipid bilayer is considered to be properly formed and havethe necessary structural and electrical characteristics, otherwise thelipid bilayer is not properly formed and an erasing electrical stimulusmay be applied to erase the lipid bilayer so the process of assemblingthe lipid bilayer on the lipid bilayer compatible surface can be startedall over again.

Furthermore, techniques for inserting a nanopore into a lipid bilayerare described herein. In one example, a solution containing nanoporeforming molecules are deposited on the lipid bilayer, an agitationstimulus is applied across the lipid bilayer to disrupt the lipidbilayer and facilitate insertion of the nanopore into the lipid bilayer.The agitation stimulus may be any kind of stimulus that can causedisruption, preferably temporary disruption, of the lipid bilayer forfacilitating nanopore insertion. It may be electrical, thermal,chemical, sound (audio), mechanical, and/or light stimuli. In oneexample, the agitation stimulus is an agitation electrical voltage levelhaving a range of 100 mV to 1.0 V for 50 ms to 1 s.

In some embodiments, the lipid bilayer or the nanopore containing lipidbilayer is damaged or destroyed accidentally, or purposefully using adestruction electrical stimulus having a range of 300 mV to 3V (or −300mV to −3 V) so that a new nanopore containing lipid bilayer can beformed over the planar solid surface. The destruction of the lipidbilayer may cause the surface underneath the lipid bilayer to oxidize orreduced. In such cases, a cleaning electrical stimulus having amagnitude of 50 mV to 300 mV may be applied to reverse the oxidation orreduction of the solid surface.

The lipid bilayer may be monitored to make sure that the desired numberof nanopore(s) has been inserted and the lipid bilayer is not damagedduring the process. In one example, a measuring electrical stimulus isapplied across the lipid bilayer and a resistance (or ionic current) ofthe lipid bilayer is measured. The magnitude of the lipid bilayerresistance indicates whether any nanopore has been inserted into thelipid bilayer, if the nanopore has been inserted, how many nanoporeshave been inserted, and if the lipid bilayer has been damaged during theprocess. If it is determined that the desired number of nanopores hasbeen inserted and the lipid bilayer has not been damaged during theprocess, the lipid bilayer may be used for characterizing moleculesusing the techniques described herein. If it is determined that nonanopore has been inserted, another agitation electrical stimulus may beapplied. If it is determined that greater than the desired number ofnanopores has been inserted or the lipid bilayer has been damaged, anerasing electrical stimulus may be applied across the lipid bilayer toerase the lipid bilayer in order to restart the process of creatinglipid bilayer and inserting nanopore.

FIG. 1 is a schematic diagram of a nanopore device 100 that may be usedto characterize a molecule as described in the examples described abovewhere the nanopore containing lipid bilayer is characterized by aresistance and capacitance. The nanopore device 100 includes a lipidbilayer 102 formed on a lipid bilayer compatible surface 104 of aconductive solid substrate 106, where the lipid bilayer compatiblesurface 104 may be isolated by lipid bilayer incompatible surfaces 105and the conductive solid substrate 106 may be electrically isolated byinsulating materials 107, and where the lipid bilayer 102 may besurrounded by amorphous lipid 103 formed on the lipid bilayerincompatible surface 105. The lipid bilayer 102 is embedded with asingle nanopore structure 108 having a nanopore 110 large enough forpassing of at least a portion of the molecule 112 being characterizedand/or small ions (e.g., Na⁺, K⁺, Ca²⁺, Cl⁻) between the two sides ofthe lipid bilayer 102. A layer of water molecules 114 may be adsorbed onthe lipid bilayer compatible surface 104 and sandwiched between thelipid bilayer 102 and the lipid bilayer compatible surface 104. Theaqueous film 114 adsorbed on the hydrophilic lipid bilayer compatiblesurface 104 may promote the ordering of lipid molecules and facilitatethe formation of lipid bilayer on the lipid bilayer compatible surface104. A sample chamber 116 containing a solution of the molecule 112 maybe provided over the lipid bilayer 102 for introducing the molecule 112for characterization. The solution may be an aqueous solution containingelectrolytes and buffered to an optimum ion concentration and maintainedat an optimum pH to keep the nanopore 110 open. The device includes apair of electrodes 118 (including a negative node 118 a and a positivenode 118 b) coupled to a variable voltage source 120 for providingelectrical stimulus (e.g., voltage bias) across the lipid bilayer andfor sensing electrical characteristics of the lipid bilayer (e.g.,resistance, capacitance, and ionic current flow). The surface of thenegative positive electrode 118 b is or forms a part of the lipidbilayer compatible surface 104. The conductive solid substrate 106 maybe coupled to or forms a part of one of the electrodes 118. The device100 may also include an electrical circuit 122 for controllingelectrical stimulation and for processing the signal detected. In someembodiments, the variable voltage source 120 is included as a part ofthe electrical circuit 122. The electrical circuitry 122 may includeamplifier, integrator, noise filter, feedback control logic, and/orvarious other components. The electrical circuitry 122 may be integratedelectrical circuitry integrated within a silicon substrate 128 and maybe further coupled to a computer processor 124 coupled to a memory 126.

The lipid bilayer compatible surface 104 can be formed from variousmaterials that are suitable for ion transduction and gas formation tofacilitate lipid bilayer formation. In some embodiments, conductive orsemi-conductive hydrophilic materials as opposed to insulatinghydrophilic materials are preferred because they may allow betterdetection of a change in the lipid bilayer electrical characteristics.Example materials include Ag—AgCl, Ag—Au alloy, Ag—Pt alloy, or dopedsilicon or other semiconductor materials.

The lipid bilayer incompatible surface 105 can be formed from variousmaterials that are not suitable for lipid bilayer formation and they aretypically hydrophobic. In some embodiments, non-conductive hydrophobicmaterials are preferred, since it electrically insulates the lipidbilayer regions in addition to separate the lipid bilayer regions fromeach other. Example lipid bilayer incompatible materials include forexample silicon nitride (e.g., Si₃N₄) and Teflon.

In one particular example, the nanopore device 100 of FIG. 1 is a alphahemolysin (αHL) nanopore device having a single αHL protein 108 embeddedin a diphytanoylphosphatidylcholine (DPhPC) lipid bilayer 102 formedover a lipid bilayer compatible silver-gold alloy surface 104 coated ona copper material 106. The lipid bilayer compatible silver-gold alloysurface 104 is isolated by lipid bilayer incompatible silicon nitridesurfaces 105, and the copper material 106 is electrically insulated bysilicon nitride materials 107. The copper 106 is coupled to electricalcircuitry 122 that is integrated in a silicon substrate 128. Asilver-silver chloride electrode placed on-chip or extending down from acover plate 130 contacts an aqueous solution containing dsDNA molecules.

The αHL nanopore is an assembly of seven individual peptides. Theentrance or vestible of the αHL nanopore is approximately 26 Å indiameter, which is wide enough to accommodate a portion of a dsDNAmolecule. From the vestible, the αHL nanopore first widens and thennarrows to a barrel having a diameter of approximately 15 Å, which iswide enough to allow a single ssDNA molecule to pass through but notwide enough to allow a dsDNA molecule to pass through. At a given time,approximately 1-20 DNA bases can occupy the barrel of the αHL nanopore.

In addition to DPhPC, the lipid bilayer of the nanopore device can beassembled from various other suitable amphiphilic materials, selectedbased on various considerations, such as the type of nanopore used, thetype of molecule being characterized, and various physical, chemicaland/or electrical characteristics of the lipid bilayer formed, such asstability and permeability, resistance, and capacitance of the lipidbilayer formed. Example amphiphilic materials include variousphospholipids such as palmitoyl-oleoyl-phosphatidyl-choline (POPC) anddioleoyl-phosphatidyl-methylester (DOPME),diphytanoylphosphatidylcholine (DPhPC) dipalmitoylphosphatidylcholine(DPPC), phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidic acid, phosphatidylinositol,phosphatidylglycerol, and sphingomyelin.

In addition to the αHL nanopore shown above, the nanopore may be ofvarious other types of nanopores. Examples include γ-hemolysin,leukocidin, melittin, and various other naturally occurring, modifiednatural, and synthetic nanopores. A suitable nanopore may be selectedbased on various characteristics of the analyte molecule such as thesize of the analyte molecule in relation to the pore size of thenanopore. For example, the αHL nanopore that has a restrictive pore sizeof approximately 15 Å. It is suitable for analyzing DNA molecules sinceit allows a single strand DNA (ssDNA) to pass through while restrictinga double strand DNA (dsDNA).

FIG. 2 is a schematic diagram of an example electrical circuit 122 of asingle cell of a nanopore array. The electrical circuit 122 is used forcontrolling the electrical stimulus applied across the lipid bilayer 102which contains a nanopore and for detecting electrical signatures orelectrical patterns of the molecule passing through the nanopore. Thethick lines represent analog signal levels and the thin lines representlogic signal levels. As shown here, the circuit 122 includes a pair ofelectrodes 118 a, 118 b placed across the nanopore containing lipidbilayer 102. The surface of the positive electrode 118 b forms the lipidbilayer compatible surface 104 and the surfaces of the adjacent siliconnitride 107 form the lipid bilayer incompatible surfaces 105. The inputvoltage applied across the lipid bilayer by the electrodes is controlledby selecting an input source from a plurality of input sources 202 atthe multiplexer 204. Each of the plurality of voltage sources canprovide DC, AC, pulse, ramp AC and/or ramp DC signals. The signal isamplified by an amplifier 206 and then compared with a set value 214 bya comparator 212, which outputs a signal when the amplified signalreaches the set value 214.

The time for the amplified signal to reach the set value 214 under aconstant input voltage can be correlated with the resistance of thelipid bilayer and the ion current passing through the lipid bilayer. Alonger time corresponds to a larger resistance and a smaller ion currentthrough the lipid bilayer. The peak to peak amplitude of the amplifiedsignal as detected by comparator 214 under a modulated input voltage(e.g., modulated with a sine wave) can be similarly correlated with thecapacitance of the lipid bilayer. A larger peak to peak amplitudecorresponds to a higher capacitance.

The circuit 122 further includes capacitor 216 for reducing noise levelsand a switch 210 for resetting the capacitor 208. A logic controller 218is provided to control the operation of the various components of thecircuit and process the signal output of the comparator.

It should be noted that the above circuit design is only an example;other suitable circuit designs may also be used for controlling theelectrical stimulus applied across the lipid bilayer and for measuringthe electrical characteristics or signatures of the surface above theelectrode, such as the electrical characteristics or signatures of thelipid suspension, lipid bilayer, nanopore containing lipid bilayer,and/or analyte molecule passing through the nanopore contained in thelipid bilayer.

FIG. 3A is a top view of a schematic diagram of an embodiment of ananopore chip 300 having an array 302 of individually addressablenanopore devices 100 having a lipid bilayer compatible surface 104isolated by lipid bilayer incompatible surfaces 105. Referring back toFIG. 1 , each nanopore device 100 is complete with a control circuit 122integrated on a silicon substrate 128. In some embodiments, side walls136 may be included to separate groups of nanopore devices 100 so thateach group may receive a different sample for characterization. Withreference to FIG. 3A, in some embodiments, the nanopore chip 300 mayinclude a cover plate 130. The nanopore chip 300 may also include aplurality of pins 304 for interfacing with a computer processor. In someembodiments, the nanopore chip 300 may be coupled to (e.g., docked to) ananopore workstation 306, which may include various components forcarrying out (e.g., automatically carrying out) the various embodimentsof the processes of the present invention, including for example analytedelivery mechanisms such as pipettes for delivering lipid suspension,analyte solution and/or other liquids, suspension or solids, roboticarms, and computer processor, and memory. FIG. 3B is a cross sectionalview of the nanopore chip 300.

FIG. 4 is a schematic diagram depicting an example process 400A forassembling a lipid bilayer on the lipid bilayer compatible surface 104.The process 400A may be carried out using the nanopore device 100 ofFIGS. 1 and 3 . FIGS. 4B, 4C, and 4D illustrate the various phases ofthe nanopore device 100 during the process.

Referring back to FIG. 4A, in this example, the lipid bilayer is adiphytanoylphosphatidylcholine (DPhPC) lipid bilayer. The lipid bilayercompatible surface 104 is an Ag—Au alloy surface isolated by one or morelipid bilayer incompatible silicon nitride surfaces. One or more stepsof the process may be automated using an electrical circuit, computerhardware and/or computer software. The top trace 402 represents theprofile of a voltage applied across the lipid bilayer. The bottom trace404 represents a resistance profile detected across the lipid bilayer.

At time t₀, an aqueous lipid suspension containing 10 mg/mL colloidaldiphytanoylphosphatidylcholine (DPhPC) dissolved in decane and 0.1 Mpotassium formate dissolved in 1 M KCl is deposited on the Ag—Au alloyelectrode surface. The lipid suspension may be deposited for exampleusing a liquid dispenser such as a pipette. In some embodiments, theliquid dispenser may be automated with various hardware (e.g., roboticarms) and software. Ag—Au alloy is hydrophilic and causes the lipidmolecules to self-organize on its surface in a way that promotes lipidbilayer formation. At time t₀-t₁, the nanopore device is in Phase I(illustrated in FIG. 4B). In Phase I, amorphous lipids 103 concentrateon the lipid bilayer incompatible surface 105 and are only barelypresent over the lipid bilayer compatible surface 104. A measuringvoltage (˜50 mV) 406 is applied to the electrode. The resistance versustime profile 408 of the electrode shows that the resistance isrelatively low (˜10 KΩ to 10 MΩ) and the electrode is shorted.

At time t₁, a bubble initiating stimulus 410 having a range of ˜1.4 V to˜3.0 V and a duration of ˜100 ms to ˜1 s is applied across theelectrode. The bubble initiating stimulus 410 causes the formate, whichwe believe is mostly present over the hydrophilic lipid bilayercompatible silver-gold alloy surface and not over the hydrophobic lipidbilayer incompatible silicon nitride surface, to decompose to formgaseous CO₂, which causes a bubble 130 to form on the solid silver-goldalloy electrode surface. The nanopore device is in Phase II (illustratedin FIG. 4C). The bubble covers the electrode and stops when it reachesthe amorphous lipid material 103 at the edge of the lipid bilayercompatible surface 104. An electrical and mechanical seal is formed overthe lipid bilayer compatible surface. The resistance versus time profile412 at time t₁-t₂ shows a dramatic increase in resistance (e.g., >10 GΩ)due to the formation of the bubble.

At time t₂-t₃ (˜100 ms to 1 s), CO₂ diffuses out of the bubble rapidly,causing the bubble to collapse and gradually form a lipid bilayer. Thenanopore device is in Phase II (illustrated in FIG. 4C) 102 over thesolid electrode surface 104. The lipid bilayer is surrounded byamorphous lipid 103 aggregated over the lipid bilayer incompatiblesilicon nitride surface 105. The resistance across the nanopore device416 under an applied measuring voltage (˜50 mV) 414 remains high due thepresence of the lipid bilayer 102.

At time t₃-t₄ (˜50 ms to 500 ms), a lipid bilayer 102 has been formedand the nanopore device is in Phase III (illustrated in FIG. 4D). Analternating current 418 is applied across the lipid bilayer to check forproper lipid bilayer resistance 420 and/or capacitance (not shown). Aproperly formed lipid bilayer with sound structural integrity isdetermined to be formed if the measured capacitance has a value greaterthan approximately a 5 fF/μm² and if the measured resistance has a valuegreater than approximately 10 GΩ. Otherwise, the lipid bilayer isdetermined to have poor structural integrity. If it is determined thatthe lipid bilayer has sound structural integrity, the nanopore device100 is ready for nanopore insertion as will be illustrated in referenceto FIG. 5 . If it is determined that the lipid bilayer has poorstructural integrity, a destruction or erasing electrical stimulus(e.g., ˜2 V) is applied across the lipid bilayer to erase the lipidbilayer. The nanopore device 100 reverts back to Phase I (illustrated inFIG. 4B).

FIG. 5A is a schematic diagram of an embodiment of a process 500 forinserting a nanopore into a lipid bilayer. The process may beimplemented using the nanopore device 100 of FIG. 1 or 3 . The one ormore steps of the process may be automated using hardware (e.g.,integrated circuit) and/or computer code. The bilayer forming process ismonitored using the nanopore device 100 of FIG. 1 . Trace A represents avoltage applied across the lipid bilayer. Trace B represents theresistance detected across the lipid bilayer. FIGS. 5B-E illustratevarious phases the nanopore device 100 is in during the process.

Referring back to FIG. 5A, at time t₀-t₁, the nanopore device includes astructurally sound lipid bilayer membrane and the nanopore device is inPhase III (illustrated in FIG. 5B). A solution containing α-hemolysin, ananopore forming peptides, is over the lipid bilayer. Applying ameasuring stimulus (e.g., ˜50 mV) 502 across the lipid bilayer returns aresistance value 504 that falls in the desired range (˜10 GΩ),indicating a lack of ionic current through the lipid bilayer.

At time t₁-t₂, an agitation electrical stimulus 506 (˜100 mV to 1.0 Vfor 50 ms to 1 s) is applied across the lipid bilayer membrane, causinga disruption in the lipid bilayer and initiating the insertion ofα-hemolysin nanopore into the lipid bilayer.

At time t₂-t₃ and immediately following the agitation electricalstimulus 506, a negative electrical stimulus 508 is applied. Thenegative pulse is intended to reverse any oxidation (e.g., oxidation ofthe electrodes) that may have been caused by accidental bursting of thelipid bilayer.

At time t₃-t₄, a measuring electrical stimulus (˜50 mV) 510 is appliedto check for proper nanopore insertion. The magnitude of the measuredresistance 512 gives an indication whether the nanopore has beeninserted, and if nanopore is inserted how many nanopores have beeninserted, and whether the lipid bilayer has been disrupted or destroyedduring the process. 512 shows an example of a drop in resistance withthe insertion of a nanopore. For example, a lipid bilayer with nonanopore inserted would have a resistance in the range of 10 GΩ, a lipidbilayer with a single nanopore inserted (Phase IV, illustrated in FIG.5C) would have a resistance in the range of 1 GM, a lipid bilayer withtwo or more nanopores inserted (Phase V illustrated in FIG. 5D) wouldhave a resistance in the range of ˜500 MΩ, and a disrupted or damagedlipid bilayer would have a resistance in the range of less thanapproximately 10 MΩ. If it is determined that no nanopore has beeninserted in the lipid bilayer, another agitation electrical stimulus maybe applied. If it is indicated that a single nanopore has been insertedand the lipid bilayer is structurally sound, the process stops and thenanopore device is ready for analyzing the analyte molecule. If it isdetected that more than one nanopore has been inserted or the lipidbilayer is disrupted, an erasing or destruction electrical stimulus(˜300 mV to 3 V) 514 can be applied to erase the lipid bilayer. Thelipid bilayer electrode is once again shorted and the nanopore device isin (Phase I, illustrated in FIG. 5E). The destruction electricalstimulus can be followed by a cleaning electrical stimulus (50 mV to 300mV) to reverse the oxidation that may have occurred on the electrodesurface due to the destruction of the lipid bilayer. The whole processof assembling lipid bilayer (e.g., FIG. 4 ) and inserting nanopore(e.g., FIG. 5 ) can be started over again.

In other techniques, nanopores are self-assembled. In these techniques,the concentration of the nanopore forming solution must be high enoughsuch that the nanopores are inserted into the lipid bilayersautomatically and without any external stimulus. Such self-assemblytechniques have a number of drawbacks. For example, if the concentrationof the nanopore forming solution is too low, then no nanopores areinserted into the lipid bilayers. If the concentration is too high, thenmultiple nanopores may be formed in a lipid bilayer, unless the nanoporeforming solution is flushed after the first nanopore is formed. Inaddition, there is little or no control of when the nanopores will beformed.

In contrast to the self-assembly techniques, the present applicationdiscloses an electroporation technique in which an agitation electricalstimulus 506 is applied across the lipid bilayer membrane to disrupt thelipid bilayer and initiate the formation of a nanopore in the lipidbilayer. The concentration of the nanopore forming solution may bereadily maintained at a sufficiently low enough level such that nonanopores are inserted automatically into the lipid bilayers. Inaddition, the agitation electrical stimulus 506 may be applied in such away that only a single nanopore is inserted into each lipid bilayer.

Electroporation allows lower amounts (lower concentration) of hemolysinprotein in the nanopore forming solution to achieve pore insertions. Insome embodiments, the concentration of pore protein is at a level suchthat when applied to an array of bilayer-covered sensors, less than 10percent of the bilayers have pores inserted in an uncontrolled manner.Using a lower concentration of pore protein has a number of advantages.For example, a user can have better control of the pore insertionbecause once a pore is inserted by electroporation, a second pore isunlikely to be inserted by itself. Using a lower concentration of poreprotein is more economical. In addition, a lower concentration of poreprotein is safer for the user to handle. For example, a highconcentration of pore protein spilled on human skin may cause a severerash, thus posing a health hazard.

If the electroporation technique described herein is not used, but asolution of protein pores is simply applied to an array of independentbilayers, it is well known to those skilled in the art that theinsertion of protein pores into lipid bilayers will follow a Poissondistribution. This independent, randomness in pore insertion over timemeans that not only pore insertions are uncontrolled but that insertionsare independent of prior events and therefore illustrates that more thanone pore can be inserted into a single bilayer. Such a random systemlimits the percentage of single pore instances that one may obtain usingconcentration of pore protein alone. Changing a nanopore system from arandom set of events with a Poisson distribution to a system withactive, deterministic pore insertion (not following the Poissondistribution) gives the new system great advantage in attaining a higherpercentage of bilayers with single pores.

The level of the agitation electrical stimulus 506 at which a nanoporeis inserted into a lipid bilayer may vary based on various factors,including salt concentration, temperature, the type of lipid membranematerial, size of the lipid bilayer, and the like. In some embodiments,about 80-90% of the nanopores are inserted when the magnitude of theagitation electrical stimulus 506 is within the range of 200 mV to 450mV. The rest of the nanopores may be inserted at lower voltage levels,e.g., 100 mV or at higher levels, e.g., 700 mV.

In some embodiments, the steps of applying the agitation electricalstimulus 506, applying the negative stimulus (reverse oxidationstimulus) 508, and applying the measuring electrical stimulus 510 fordetecting whether a nanopore has been properly formed are iterated in aloop, until a nanopore is finally formed in the lipid bilayer.Initially, the magnitude of the agitation electrical stimulus 506 is setat a lower level. The magnitude of the agitation electrical stimulus 506is then gradually increased after each iteration. For example, theinitial agitation electrical stimulus 506 may be set at a lower level,such as 60 mV. After each iteration, if a nanopore is still not properlyinserted, then the agitation electrical stimulus 506 is adjusted by asmall increment, e.g., 2 mV. This process may be repeated until ananopore is finally inserted into the lipid bilayer, or until the lipidbilayer is damaged or erased by the agitation electrical stimulus 506,or until the agitation electrical stimulus 506 has reached apredetermined maximum threshold, e.g., 700 mV. By gradually increasingthe agitation electrical stimulus 506 level (e.g., from 60 mV to 700 mV)as describe above, a higher percentage of the cells of the nanopore chipwill have one nanopore properly inserted into a lipid bilayer.

In FIG. 5A, agitation electrical stimulus 506 is shown as a positivevoltage. However, a negative agitation electrical stimulus may be usedin some embodiments as well. When a negative agitation electricalstimulus is used, a positive pulse may be used to reverse any oxidation(e.g., oxidation of the electrodes).

Because individual pore protein mixes can have smaller or largerpercentages of well-formed, active pore molecules, the desiredconcentration to use for different batches of pore protein varies. Insome embodiments, a particular pore protein mix is first tried on a chipto see how active it is and then the concentration is adjusted untilapplication of the mix will put only a few (e.g., 0-10) pores into anarray of 264 bilayer covered electrodes without any stimulation. Ingeneral, a concentration may be chosen that results in less than about10 percent pore formation un-stimulated. In some embodiments, aconcentration may be chosen that results in less than about 30 percentpore formation un-stimulated. At such a concentration level, aninsignificant number of pores will be inserted when the mix is simplyleft on the array with no stimulation. At this low concentration,electroporation techniques will insert pores between −100 mV and −600mV. Positive pulses can be used as well.

In some embodiments, applying concentrations of alpha-hemolysin or MspApores in the range of 0.1 ng/mL (nanogram) to 2 μg/mL (microgram) ofpore protein will typically result in the preferred condition of havingonly a few pores inserted in a field of 264 bilayer covered electrodesunaided. The exact concentration used may be determined aftercalibration on a chip and prior to distribution of the protein pore mixto researchers. In such a case, the resulting protein mix is diluted tothe optimal level and then stored for future use and distribution toresearchers where further calibration is not necessary.

The amount or concentration needed to reach the desired state describedabove varies with salt concentration, temperature, pressure, and thedimensions of the bilayers covering the electrodes. Higher temperaturesor pressures require lower pore concentration. Larger bilayer diametersalso require a lower pore concentration to successfully implement anelectroporation scheme for directed, controlled pore insertion. The poreconcentrations above take into account possible variations for saltconcentrations (KCl or NaCl) (e.g., from 50 mM to 1M), temperatures(e.g., from 0 degree Celsius to 25 degree Celsius), pressures (e.g.,normal barometric pressure at sea level), and bilayer diameters (e.g.,from approximately 5 μm to 250 μm). The technique described above oftesting a given mix or representative mix allows for these variances tobe taken into account.

FIG. 6A is a schematic diagram illustrating an embodiment of a process600 for manipulating, detecting, correlating, characterizing, analyzingand/or sequencing a molecule in a nanopore using a nanopore device. Oneor more steps of the process may be automated via hardware (e.g.,integrated circuit) and/or execution of a computer code. In the exampleillustrated, a dsDNA molecule is characterized using a αHL nanoporeinserted in a lipid bilayer such as a DPhPC lipid bilayer formed on thenanopore device as illustrated in FIG. 1 or 3 . FIGS. 6B-C illustratethe various phases the nanopore device is in during the process.

Referring back to FIG. 6A, Trace A represents a voltage applied acrossthe nanopore containing lipid bilayer. Trace B represents the resistancedetected across the nanopore containing lipid bilayer. At time t₀, ananalyte solution containing a double stranded DNA (dsDNA) molecule ispresented to the lipid bilayer, by for example depositing the analytesolution adjacent to the lipid bilayer. The analyte solution in thisexample is an aqueous solution containing the analyte molecule and smallelectrolytes (e.g., Na⁺, K⁺, Ca²⁺, Cl⁻) that is buffered to anappropriate pH 7.5 to 8.0. The nanopore has an open channel and theresistance of the nanopore containing lipid bilayer has a resistance ofapproximately 1 GΩ (Phase IV, illustrated in FIG. 6A)

At time t₀-t₁, an acquiring electrical stimulus (˜100 mV to 400 mV) 602is applied across the lipid bilayer of the nanopore device, causing asingle dsDNA molecule to be captured in the nanopore (Phase V,illustrated in FIG. 6A). The resistance versus time profile shows asharp increase in resistance 604 to 6 GΩ which corresponds to anobstructed pore state (Phase V, illustrated in FIG. 6C) where thenanopore is partially blocked by a dsDNA molecule.

At time t₁-t₂, the sharp increase in resistance 604 triggers a controlmechanism (e.g., the feedback control mechanism in circuit 122 of FIG. 2) to lower the electrical stimulus to a holding electrical stimulus (˜50mV to 150 mV) 608 with a fast response time (e.g., <10 mS) 606 in orderto hold the dsDNA in the nanopore for detection, characterization and/oranalysis. The short response time allows the analyte molecule to betrapped in the nanopore for characterization rather than passing throughthe nanopore and exiting through the other end.

At time t₂-t₃, the dsDNA molecule is held in the nanopore with theholding electrical stimulus, a first frame (f₁) of resistance versustime profile is recorded.

Subsequently from t₃-t₇, multiple series of variable progressionelectrical stimuli 609 are applied to the DNA molecule trapped in thenanopore, where each series of the variable progression electricalstimuli 610 comprises successively higher or more intense electricalpulses 613. As illustrated, each of the electrical pulses 613 comprisesa ramp-up phase 615, a ramp-down phase 617, resembling a reversed “V”and having a range of approximately 100 mV to 200 mV. Each of theelectrical pulses 613 is followed by a hold phase 619. As illustrated,the slope of the initial ramp-up phase 615 is steeper than the slope ofthe subsequent ramp-down phase 617. Each series of electrical pulses 610may result in a frame (e.g., 1 to 20 base pairs) of the dsDNA moleculeto be unzipped and the single strand of the unzipped dsDNA frame pulledthrough the nanopore under the applied progression electrical stimulus.The electrical pattern or signature of the frame of molecule is measuredduring each of the hold phases 619. The details are as follows:

At time t₃-t₄, a series of successively higher progression electricalstimulus (e.g., asymmetric electrical pulses) 610 is applied across thelipid bilayer to drive the dsDNA through the nanopore. After eachelectrical pulse 613, the resistance versus time profile is monitoredduring the hold phase 619 immediately following the electrical pulse613. If the resistance versus time profile detected is the same as thatof the previous frame f₁, it indicates that the electrical stimuluslevel is not high enough to drive the DNA molecule through the nanopore,and a higher electrical stimulus level is applied. The process ofsuccessively applying a higher electrical stimulus level is repeateduntil a different resistance versus time profile indicates that a newframe f₂ has been obtained and the new frame is recorded.

At time t₄-t₅, the previous process of applying successively higherprogression electrical stimulus to pull the DNA molecule is repeateduntil a new frame f₃ is obtained.

At time t₅-t₆, the previous process of applying variable andsuccessively higher progression electrical stimulus to pull the DNAmolecule is repeated to obtain a new frame f₄ is recorded.

At time t₆-t₇, the previous process of applying successively higherprogression electrical stimulus is repeated to obtain a new frame f₅.This process of applying successively higher progression electricalstimulus to obtain a new frame may be repeated.

At time beyond t₇, the resistance versus time profile may reach a levelthat corresponds to an open state for the nanopore (Phase IV,illustrated in FIG. 6B) 612. This indicates that the DNA molecule hasescaped the nanopore and the flow of ions in the nanopore is unhinderedby DNA molecule.

Each of the various frames (f₁ to f₅) corresponds to a resistanceinformation when a particular region of the DNA molecule is lodged inthe narrow passage of the nanopore. The various frames, separately or incombination, can be used to elucidate, detect, correlate, determine,characterize, sequence and/or discriminate various structural andchemical features of the analyte molecule as it traverses the nanopore.In some embodiments, one or more frames of the molecule may overlap. Theoverlapping of the sampling frames may allow for a more accuratecharacterization of the DNA molecule. For example, a single strand of adsDNA molecule is threaded through the nanopore and the ssDNA has asequence of 5′TGACTCATTAGCGAGG . . . 3′. The first frame of the moleculeis the electrical signature detected for the segment TGACT, the secondframe is the electrical signature detected for ACTCA, the third frame isthe electrical signature detected for TCATT, and the fourth frame is theelectrical signature detected for ATTAG, and so on and so forth. Theelectrical signatures of the various overlapping frames can be combinedand deconvolved to generate a more accurate electrical signature of themolecule.

Although in this example, reversed “V” shaped progression electricalstimuli pulses 613 with an initial ramp-up phase 615 and a subsequentramp-down phase 617 are used, other types of the progression electricalstimuli pulses may be used. In some embodiments, the progressionelectrical stimuli pulses may resemble a square wave (as illustrated inFIG. 7A), a smooth wave (as illustrated in FIG. 7B), or a reversed “U”with a flat center (as illustrated in FIG. 7C). In some embodiments, theprogression electrical stimulus does not have the ramp-up phase 615 andthe ramp-down phase 617, for example the progression electrical stimulusincludes a steady constant progression electrical stimulus 610 (asillustrated in FIG. 7D).

Although in this example, a hold phase 619 follows each of theprogression electrical stimuli pulses 613 and the electrical signatureof the molecule is measured during the each of the hold phases 619, inother embodiments the hold phases 619 may be eliminated and theelectrical signature of the molecule may be measured (e.g.,continuously) while the progression electrical stimuli are applied andwhile the molecule is moving through the nanopore under the appliedprogression electrical stimuli. In one example, reversed “V” shapedprogression electrical stimuli pulses 613 are applied without the holdphases 619, the electrical signature of the molecule is measured as theprogression electrical stimulus is ramped up and ramped down (e.g.,applied voltage at the electrode is ramping up or down). In suchinstances, the electrical signature of the molecule (e.g., resistanceprofile of the molecule) can be determined as a function of varyingprogression electrical stimulus level (e.g., varying voltage level) andsuch information can be used to differentiate different molecules (e.g.,different DNA frames) being characterized. In another example, aconstant progression electrical stimulus is applied without a hold phaseand the electrical signature of the molecule is measured as the constantprogression electrical stimulus is applied and while the molecule ismoving through the nanopore under the constant progression electricalstimulus.

As discussed previously FIGS. 7A-D illustrate various embodiments of theprogression electrical stimulus in addition to the reversed “V” shapedprogression electrical stimulus.

FIG. 8 is a schematic diagram illustrating an embodiment of a process800 for reversing the progression of a molecule in a nanopore of ananopore device. In the example as illustrated, a dsDNA is analyzedusing a αHL nanopore. Constant progression electrical stimuli andreverse progression electrical stimuli are used, and the electricalsignature of the molecule is recorded continuously while the constantprogression electrical stimuli and reverse progression electricalstimuli are applied and while the molecule is moving through thenanopore.

Although constant progression electrical stimuli are used in thisexample, various other types of progression electrical stimulus can beused. Examples of the various progression electrical stimulus areillustrated in FIGS. 6 and 8 . Although constant reverse progressionelectrical stimuli are used in this example, various other types ofreverse progression electrical stimulus can be used. The reverseprogression electrical stimulus can include a ramp-up and/or a ramp-downand can include a smooth, square, “V”, and/or “U” shaped profile similarto the progression electrical stimulus.

Trace A represents a voltage applied across the nanopore containinglipid bilayer. Trace B represents the resistance detected across thelipid nanopore containing bilayer. One or more steps of the process maybe automated using hardware (e.g., integrated circuit) and/or executionof computer code.

At time t₀-t₁, a progression electrical stimulus 802 is applied acrossthe lipid bilayer of the nanopore device, causing the dsDNA molecule tomove in the direction of the applied electrical force 805 (Phase V,illustrated in FIG. 8B) as a resistance versus time profile 804 of thelipid bilayer is recorded.

At time t₁-t₂, a reverse progression electrical stimulus 806 is appliedacross the lipid bilayer. In this example, the reverse progressionelectrical stimulus 806 is an applied voltage level having a range of˜−50 mV to 100 mV. The natural tendency for the ssDNA molecule tore-associate to form a dsDNA drives the DNA molecule in the reversedirection 807 (Phase VI, illustrated in FIG. 8C). As the DNA molecule ispushed back through the nanopore in the reverse direction 807, ssDNAre-associates to form a dsDNA.

At time beyond t₂, a progression electrical stimulus 810 is againapplied across the lipid bilayer, resuming the forward progression ofthe DNA molecule (Phase V, illustrated in FIG. 8B).

FIG. 9 is example resistance versus time profile 902 detected as asingle strand of a dsDNA molecule was threaded through a αHL nanoporeusing the techniques described herein. In the example shown, a constantprogression electrical stimulus is applied to nanopore containing lipidbilayer, the electrical signature of the DNA molecule trapped in thenanopore is recorded continuously while the constant progressionelectrical stimulus is applied and while the DNA molecule is movingthrough the nanopore. The base sequence of the DNA molecule can bedetermined by comparing the detected resistance profile with theresistance profile(s) of known DNA sequence(s). For example, the basesequence of the DNA molecule may be determined to be that of a known DNAmolecule if the resistance versus time profiles match. The variousfeatures of the profile, such as amplitude, frequency, edge rise (e.g.,edge rise time), and/or edge fall (e.g., edge fall time) may be used toidentify a particular DNA sequence.

Although the foregoing embodiments have been described in some detailfor purposes of clarity of understanding, the invention is not limitedto the details provided. There are many alternative ways of implementingthe invention. The disclosed embodiments are illustrative and notrestrictive.

Although electrical signatures expressed in terms of resistance versustime profile in the various embodiments described herein, it should benoted that the electrical signatures can also be expressed in terms ofvoltage versus time profile and/or current versus time profile in otherembodiments. It should also be noted that an electrical property can bedirectly measured or indirectly measured. For example, resistance can bedirectly measured or indirectly measured by the voltage and/or thecurrent, and current can be measured directly or indirectly measured byresistance and/or voltage. All ranges of electrical stimuli are givenfor a particular example nanopore system described herein. In othernanopore systems where chemistry is different, different ranges ofelectrical stimuli may apply.

What is claimed is:
 1. A method for forming a nanopore in a membrane,the method comprising: depositing a nanopore forming solution over amembrane; detecting whether a nanopore has been formed in the membrane;and in the event that a nanopore is not detected, iteratively initiatingformation of a nanopore in the membrane by increasing the amplitude ofthe voltage stimulus waveform applied to the membrane and detectingwhether a nanopore has been formed in the membrane, wherein the step ofiteratively initiating formation of a nanopore in the membrane isrepeated until formation of a nanopore is detected.
 2. The method ofclaim 1, further comprising applying a measuring voltage stimulus to themembrane to determine whether the nanopore has been formed in themembrane based on a measurement in response to the measuring voltagestimulus, wherein an absolute magnitude of the voltage stimulus waveformamplitude is different from an absolute magnitude of the measuringvoltage stimulus amplitude.
 3. The method of claim 1, wherein the stepof iteratively initiating formation of a nanopore in the membrane isrepeated until damage to the membrane is detected.
 4. The method ofclaim 1, wherein the step of iteratively initiating formation of ananopore in the membrane is repeated until the voltage stimulus waveformhas reached a predetermined maximum threshold.
 5. The method of claim 1,wherein the step of detecting whether a nanopore has been formed in themembrane comprises detecting a change in an electrical property of themembrane resulting from the formation of a nanopore in the membrane. 6.The method of claim 5, wherein the step of detecting a change in themembrane electrical property comprises detecting a change in aresistance of the membrane.
 7. The method of claim 6, wherein the stepof detecting whether a nanopore has been formed in the membranecomprises determining a number of nanopores formed in the membrane basedon a size of change in the membrane electrical property.
 8. The methodof claim 7, further comprising applying an erasing voltage stimuluswaveform level to erase the membrane when it is determined that morethan one nanopore is formed in the membrane.
 9. The method of claim 1,further comprising applying a reverse oxidation stimulus level to themembrane after the voltage stimulus waveform is applied.
 10. The methodof claim 9, wherein the voltage stimulus waveform and the reverseoxidation stimulus level comprise a positive voltage stimulus level anda negative voltage stimulus level.